Microgametophytic Selection as a Way to Improve Drought Tolerance in Cultivated Plants

Abstract

In the current chapter, using different agricultural crops as an example, the effectiveness of pollen selection techniques for drought and heat resistance are demonstrated, as well as methods to evaluate plant drought tolerance by its male gametophyte. Germination of pollen from F1 sunflower hybrid on the stigmas moistened with an osmotic resulted in drought resistance improvement of F2 sporophytic generation, increasing the number of drought tolerant genotypes. Heating sunflower pollen increased both the plant adaptability to drought in dry field conditions and germination of seeds under osmotic stress. Pollination with heated pollen created opportunities to increase the share of drought resistant genotypes in the progeny of oil flax sporophytes. In interspecific tomato hybrids pollen treatment with high temperature was accompanied by a predominant elimination of unstable to various stresses pollen grains with cultivated species alleles in favor of wild ones. The high-temperature impact on the heterogeneous pollen population increased the proportion of drought-resistant genotypes in the sporophyte population and changed the Mendelian segregation ratios for a number of marker genes in maize. The genes were revealed, which influence drought resistance or are linked to the genes responsible for tolerance of pollen and plants to water stress in some crops.

1. Introduction

Gametophytic selection is the selection of genotypes in the sexual (haploid) generation of a plant’s life cycle. Gametophytic selection is based on the different selective value of gametes, due to their genetic diversity. In this case, both micro and macrogametophytes can be subject to selection.

Selection in the haploid phase is widespread in higher plants. However, it was only recently shown that a significant part of the plant genome is expressed in the haploid phase and that a fairly large part of it is common for the sporophyte. The extensive overlap between the gametophytic and sporophytic phases constitutes the biological basis for the sporophyte response to gametophyte selection. Given that selection at the haploid level and, especially, in the male gametophytic generation, is capable to cause a significant shift in the genetic structure of populations in a short time, it can be actively involved in the breeding practice to search for valuable genotypes.

If each phase of a plant’s life cycle had its own set of expressed genes, then selection at the gametophytic level would only lead to changes in the next gametophytic generation and would not affect the sporophyte. However, the results of numerous studies on the influence of gametophytic selection on sporophytic offspring [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14] indicate that there is a significant degree of overlap between the two phases of the life cycle. It is believed that approximately 60% of the structural genes expressed in pollen are also expressed in the sporophyte [15, 16].

Selection can operate in both the male and the female gametophytic generations. However, given the large size of the population of male gametes, the relative independence of pollen grains from the mother plant, the possibility of direct impact on them by environmental factors, the competition of many gametophytes within one style, male gametophytic selection, in comparison with female selection, is considered more effective. It is often compared to selection in microorganisms, given the large population size and the haploid state of the genome [3, 16, 17].

Considering the significant degree of genetic overlap of both phases of the plant life cycle, it is believed that the efficiency of gametophyte selection, especially pollen, may be higher than that of sporophyte [16]. This is explained by the fact that, firstly, the size of the population of male gametophytes is much larger than the size of the population of sporophytes. This makes it possible to apply high selection pressure. Secondly, it is much more difficult to obtain the desired gene combination at the sporophyte stage than at the gametophyte stage as the sporophyte of higher plants contains a double set of genes compared to the gametophyte. Because of this, the probability of selecting a complex combination of alleles is higher in the gametophytic generation. And thirdly, the haploid state of the gametophyte allows direct access to the recessive alleles.

2. Microgametophytic selection for drought tolerance in cultivated plants

2.1 Microgametophytic selection for drought tolerance in sunflower

Despite the fact that the sunflower is a mesophyte, it is very demanding to the level of available soil and air humidity. That is why the yield and efficiency of its growth are limited by moisture provided to the plants. In sunflowers the yield losses may reach 50% because of drought [18]. It should be noted that drought is usually accompanied by temperature increase which invokes damage to the plants and decreases the yield as well. Therefore, enhancing the drought and heat tolerance of the plants is an important direction of modern sunflower breeding.

2.1.1 Selection of drought tolerant genotypes during pollen germination on the stigmas in vivo

Undoubtedly, successful selection for any trait is possible only if there is a genetic variability for this trait. This also applies to the trait of drought tolerance in sunflowers. Therefore, in order to prove the effectiveness of microgametophytic selection for drought resistance, genetic variability for this trait at the level of the male gametophyte (pollen) was created artificially. For this, F₁ hybrid was obtained from the crossing of inbred sunflower lines with contrasting drought resistance which was determined by the relative length and dry weight of germinal roots after seed germination in sucrose solution. At the same time, it was assumed that those inbred lines were contrasting at the level of male gametophyte.

To carry out microgametophytic selection for drought resistance, a hybrid was taken obtained from crossing the morphological mutants of ZL 9 and ZL 102 sunflower lines developed at the Institute of Oilseed Crops of NAAS (Zaporozhye, Ukraine). In addition to different resistance to drought, one mutant line was characterized by chlorophyll deficiency of the “virescent” type at the top of the seedling, and the other one had leaves with fan venation. Both mutant traits are easily identified at the seedling stage. These inherited mutant traits were the result of our long-term research on chemical mutagenesis in sunflower [19, 20]. The presence of such “marker” traits in the parental components of the F₁ hybrid made it possible to unambiguously characterize the genetic structure of the F₂ segregating population.

The modified technique, proposed by Patil et al. for sorghum, was taken into account to select male gametophytes resistant to lack of moisture in vivo during pollen germination and pollen tube growth [21]. In the experiment, the stigmas of emasculated inflorescences were moistened with 10% of polyethylene glycol (PEG) 6000 solution. In this case, PEG 6000 solution was used as an osmotic selective barrier for pollen. After a little drying of the stigmas with PEG 6000 solution, pre-emasculated inflorescences of the F₁ hybrid were pollinated with fresh pollen. The inflorescences moistened with distilled water were used for pollination in the control. One cm³ of pollen was taken for pollination of each head.

Table 1 shows the percentages of germination in 10% PEG 6000 solution of F₂ seeds [22], obtained during pollen germination of F₁ hybrid (ZL102 “virescent” x ZL9 “fan venation” crossing combination) in the presence and absence of osmotic on the stigmas of the hybrid plant.

Pollen treatment	F2 seeds		Germination, %
	Total	Germinated
Germination on the stigmas moistened with distilled water (control)	156	30	19.2
Germination on the stigmas moistened with 10% solution of PEG 6000	154	106	68.8***

Table 1.

Influence of pollen germination of F₁ hybrid in vivo in the presence of osmotic on germination of F₂ seeds in the medium with osmotic in sunflower.

Note: ***differences from the control are significant at the 0.001 level of probability.

As can be seen from the data presented in Table 1, the percentage of F₂ seed germination under conditions of osmotic stress was significantly higher in the experimental F₂ population, which was obtained after pollination with fresh pollen of the inflorescences moistened with 10% solution of PEG 6000. These results may indicate that such manipulation with a heterogeneous population of pollen from F₁ hybrid was effective and resulted in drought resistance improvement of F₂ sporophytic population, increasing there the number of drought-tolerant genotypes [23].

Table 2 shows the genetic structure of F₂ populations for “virescent” marker-trait. Change in the segregation for this marker-trait may indicate that the gene or genes, which determine the tested marker-trait, directly affect the drought tolerance of plants or are linked to the genes, which determine the plant resistance to osmotic stress and dry environmental conditions [24].

Pollen treatment	F2 phenotypes			Segregation Ratio
	Normal plants	Plants with marker-trait	Total
Germination on the stigmas moistened with distilled water (control)	198	42	240	4.7:1
Germination on the stigmas moistened with PEG 6000	366	120	486	3.0:1**

Table 2.

Influence of pollen germination of F₁ sunflower hybrid in vivo in the presence of osmotic on segregation ratio in F₂ generation for “virescent” marker-trait.

Note: χ²_0.01(df = 1) = 6.64. **differences from the control are significant at the 0.01 level of probability.

The results of marker analysis for «virescent» trait showed the modification in segregation for the experimental F₂ population, which was obtained after germination of F₁ hybrid pollen on the stigmas moistened with osmotic, compared to the control. This modification resulted in a significant increase in the number of plants with the “virescent” trait. As for “fan venation”, differences in segregation ratio for this trait were not revealed. Therefore, we can state the fact of the shift in the genetic structure of F₂ populations after carrying out gametophytic selection for drought tolerance which favored the survival of gametes with the “virescent” marker-trait and, at the same time, the elimination of gametes with an alternative trait.

In general, it must be admitted that pollination with a heterogeneous pollen population of stigmas moistened with the osmotically active substance can increase the drought resistance of F₂ sporophytic populations and modify their genetic structure for some marker traits. Also, it should be noted, that the simultaneous rise in the drought resistance and in the number of plants with the “virescent” marker-trait in F₂ population indicates that the gene or genes, which determine the “virescent” trait, directly influenced the drought resistance or were linked to the genes responsible for tolerance of pollen and plants to water stress.

2.1.2 Pollen heating as a way of selection drought tolerant genotypes

Treatment of pollen with high-temperature can also be an effective tool for breeders to overcome environmental stresses. Studies on the influence of F₁ pollen heating on the genetic structure of F₂ sporophytes showed that this technique can not only change the heat resistance of the segregating populations but its drought resistance as well. To estimate the effectiveness of pollen treatment for drought resistance improvement, comparisons were performed in the dry year 2013 on the number of plants that are able to bloom under such conditions. F₂ plants obtained from pollination with fresh or heated pollen from F₁ hybrids were analyzed.

Table 3 shows that in the year 2013 the amount of rainfall during the two months of vegetation before flowering was only about 60% of the long-term average. Before sowing in April and early May, there was almost no rainfall, while the average daily temperature was significantly higher than the average long-term data. This suggests that for cultivated sunflowers the environmental field conditions during the season of 2013 were arid, at the initial stages of growth and development especially.

Weather Parameter	April		May		June
	Temperature, °C	Rainfall, mm	Temperature, °C	Rainfall, mm	Temperature, °C	Rainfall, mm
Average daily temperature	13.2	—	23.1	—	24.7	—
Total rainfall	—	8.0	—	29.0	—	31.5
Average long-term rates	8.5	35	16.0	40	19.4	62

Table 3.

Weather conditions in Zaporozhye region (Ukraine) before and after sowing of sunflower, 2013.

F₁ hybrids were grown in the field conditions during the year 2012 to obtain F₂ seeds by self-pollination. Before flowering, inflorescences of F₁ hybrids were isolated. One set of these plants was emasculated within 1–5 days for artificial pollination. The other set was grown without emasculation for collecting fresh pollen. Freshly collected pollen was immediately (in 5–10 min after collection) transferred to the laboratory, placed in parchment packets in a layer of 2–3 mm thick, and heated at the temperature of 60°C using an air bath oven for a period of 1 h and then used to self-pollinate the emasculated F₁ plants. At the same time, freshly collected pollen without temperature treatment was used to control pollinations. One cm³ of pollen was taken for pollination of each head.

A viability test based on pollen germination on an artificial nutrient medium [25] demonstrates that the heating technique can significantly reduce the percentage of pollen germination on the artificial media. Thus, pollen treatment for 1 h at 60° C reduces the number of germinated pollen grains by almost 4 times, and treatment for 3 h—by more than 20 times, while the germination of pollen drops down to 1%.

Data on the influence of pollen heating in F₁ hybrids on the adaptability to drought in the F₂ resulting sporophytic offspring are presented in Table 4. As follows from the table, the number of flowering plants was significantly higher in F₂ populations obtained after 1-h pollen treatment than in the control. These facts indicate that gametophytic selection for heat tolerance increases the adaptability of the F₂ populations to drought stress [26].

Treatment	Number of F2 seeds	Number of F2 flowering plants	Frequency of F2 plants, %
F2 ZL9 mutant x ZL 95 mutant
Fresh pollen	137	81	59.1 ± 4.20
Pollen heated at 60°C/1 h	540	395	73.1 ± 1.91**
F2 ZL9 mutant x ZL 95 mutant
Fresh pollen	250	167	66.8 ± 2.98
Pollen heated at 60°C/1 h	514	469	91.2 ± 1.25***

Table 4.

Influence of pollen heating in F₁ sunflower hybrids on number of flowering plants in F₂ population.

Notes: **, *** the differences from the control are significant at the 0.01 and 0.001 levels of probability, respectively.

In our earlier studies, we tested the selective effect of different high temperatures on the heterogeneous pollen populations of some interspecific sunflower hybrids [27]. The results showed that pollen heating at 40°C for 3 h did not cause the selective elimination of haploid genotypes. Only the use of a higher temperature for pollen treatment led to the shift in the genetic structure of the sporophytic population. As it turned out, pollen heating at the temperature of 60°C for 1 h was already effective.

Tolerance to drought of F₂ sporophytes after heating pollen of F₁ hybrids was also assessed by the seed germination in 15% sucrose or 20% PEG solutions. The F₂ seeds were germinated at the temperature of 25°C for a period of 4 days (sucrose) and 3 days (PEG 6000). After that, the percentage of seed germination was calculated (Table 5).

Treatment	Number of F2 seeds		Germination, %
	Total	Germinated
F2 ZL95 mutant × ZL9 mutant (15% sucrose as osmotic)
Fresh pollen	480	43	9.0 ± 1.31
Pollen heated at 60°C/1 h	806	454	56.3 ± 1.75***
F2 ZL102 mutant × ZL9 mutant (20% PEG 6000 as osmotic)
Fresh pollen	156	30	19.2 ± 3.15
Pollen heated at 60°C/1 h	165	97	58.8 ± 3.83***

Table 5.

Influence of pollen heating in F₁ hybrids on seed germination of the F₂ offspring in an osmotic solution in sunflower.

Note: *** Differences from the control are significant at the 0.001 level of probability.

As can be seen from Table 5, the heating of F₁ pollen increased the drought resistance of F₂ sporophytic population estimated by seed germination in a 15% sucrose solution or in 20% PEG 6000 solution. Germination in the osmotic of F₂ seeds that were developed after pollen heating was more than 3–6 times higher compared with the F₂ seeds resulting from the pollination of F₁ hybrids with fresh pollen.

These facts reveal that gametophytic selection for heat tolerance increases both the adaptability to the drought of the F₂ populations in dry field conditions and the germination of F₂ seeds in the conditions of osmotic stress. Thus, during the pollen heating, we observe the indirect selection that increases drought resistance of sporophytic offspring. It can be reasonably assumed that the genes which define sunflower drought resistance are linked to the genes determining heat resistance.

2.2 Microgametophytic selection for drought tolerance in oil flax

Under conditions of prolonged exposure to high temperatures and insufficient moisture supply, typical for the south of Ukraine, oil flax often reduces its potential productivity. The above makes the development of drought-resistant varieties of this crop urgent. The significant differences between flax genotypes insensitivity to elevated temperatures at the stage of the mature pollen grain and the discovered positive relationship between resistance to high temperatures of microgametophytes and drought tolerance of sporophytes, which we established, provide an opportunity for selection of drought-resistant genotypes at the microgametophytic level [10, 25].

Intervarietal F₁ hybrids K7487 × K7734 and K7734 × K7487 were used as the starting material, the parental lines of which significantly differed in their resistance to drought. Pollen was collected in the morning hours. Using a dissecting needle, the pollen from the anthers was shaken out onto a glass slide and after that, it was evenly distributed over the surface of the glass in a monolayer. Then glasses with pollen were placed in a thermostat and heated at 35°C for 1–2 hours. Pollen heating at a higher temperature is undesirable, since treatment at 45°C even for a short time results in a complete loss of pollen viability.

After heating, pollen was applied to the stigmas of one of the parental components of the hybrid. The fact of selection in the experiment was evaluated by the setting of seeds and capsules in comparison with pollination with freshly collected pollen. The selection efficiency was determined by the germinating BC₁ seeds in an osmotic medium (Table 6).

Pollen treatment	Germination, %	Root length, mm
BC1 K7487 x (K7487 × K7734)
Fresh pollen	5.0 ± 2.18	1.8 ± 0.40
Pollen heated at 35°C/1 h	19.7 ± 3.39***	1.7 ± 0.17
Pollen heated at 35°C/2 h	34.7 ± 2.57***	2.8 ± 0.28**
BC1 K7487 × (K7734 × K7487)
Fresh pollen	5.2 ± 2.11	1.2 ± 0.20
Pollen heated at 35°C/1 h	17.9 ± 3.18***	1.9 ± 0.25
Pollen heated at 35°C/2 h	24.2 ± 3.91***	5.7 ± 1.24**

Table 6.

Influence of pollen heating in F₁ hybrids on seed germination of BC₁ offspring on osmotic in oil flax.

Note: **, *** Differences from the control are significant at the 0.01 and 0.001 levels of probability.

As a result of pollination with heated pollen, the setting of bolls and seeds significantly (almost by 2–3 times) decreased. Even after 1 h treatment of pollen, these parameters were significantly lower than in the control.

The heating of pollen from hybrid plants influenced both the percentage of seed germination and the length of roots developed in a medium with an osmotic. At the same time, the maximum differences from the control were observed when the pollen was heated for 120 min. Thus, the selection of pollen resistant to high temperatures in F₁ makes it possible to significantly increase the percentage of drought-resistant genotypes in the progeny of sporophytes obtained after pollination with the heated pollen. With the help of this methodological technique flax variety “Pivdenna nich” was developed which is a National Standard in Ukraine, and is perfectly adapted to the arid conditions of the south of the country.

2.3 Assessment of drought tolerance in castor bean by pollen

The climatic conditions of the south of Ukraine often have an adverse effect on the growth and development of castor bean plants, which are relatively unstable to drought and high temperatures. Therefore, the search for drought-resistant genotypes is the key aspect of plant breeding programs, which aim to obtain high-yielding varieties of this crop.

Express methods based on the analysis of pollen can be classified as promising in evaluating sporophytes for resistance to abiotic environmental factors, which is due not only to the experimentally revealed expression of a large number of genes at both stages of the plant life cycle but also to the ease of manipulation with such substance as pollen.

The correlation coefficient we established between the drought resistance of the sporophyte and the resistance of the male gametophyte to the action of high temperatures in the range of 0.72–0.89 opens the way to distinguish drought-resistant castor bean genotypes at the pollen level. An indicator of pollen resistance to elevated temperatures is the degree to which the percentage of pollen germination or the length of the pollen tube decreases after a stress factor is applied to mature pollen grains. When treating pollen of drought-resistant genotypes at the temperature of 40°C for 1 h, the degree of decrease in pollen germination percentage, as a rule, does not exceed 25%, while the length of a pollen tube may practically be of the same size.

As an example, data on the heat resistance of pollen can be provided for two genotypes of castor beans contrasting in resistance to drought (Khortytskaya 1 variety—tolerant to drought, K161 line—not tolerant to drought). Drought resistance of genotypes at the sporophyte stage was evaluated according to the method based on determining the osmotic pressure of cell sap by a refractometric method (−1.95 for Khortytskaya 1 variety, −1.0 for K161 line). In this case, the highest negative values of the water potential indicate the maximum ability of tissues to maintain it better, that is, to prevent the adverse effects of drought.

To estimate the tolerance of male gametophyte to high temperatures, pollen was subjected to temperature treatment at 40°C for 1 h, and then germinated in vitro. The tolerance of male gametophyte was assessed by the degree of decrease in pollen germination and pollen tube length in comparison with the control (pollen not subjected to heat processing).

As it turned out, in those genotypes whose sporophytes are characterized by high drought resistance, the degree of decrease in the germination of pollen after temperature treatment is minimal, while not drought-resistant lines show more significant inhibition of pollen germination when exposed to high temperatures (in Khortytskaya 1–18.8%, in K161 line—63.7%). The thermal treatment used also revealed significant differences between the accessions according to their ability to develop a long pollen tube. After heating pollen, in genotypes that were assessed as drought-resistant at the sporophyte stage, such stress caused a less significant decrease in the length of the pollen tubes than in not drought-resistant genotypes. In view of the above, a method of pollen evaluation can be proposed for screening drought-resistant castor bean samples.

2.4 Heat tolerance of tomato pollen and possibility of gametophytic selection for resistance to temperature stress

It is important in tomato breeding, especially for greenhouse cultivation, to keep in mind the heat resistance of pollen and to develop varieties and hybrids with this property. There are also known studies by G.I. Tarakanov et al. [28], who, taking into account the unequal value of different pollen grains in terms of heat resistance, used the most resistant gametes to obtain offspring. As a result of single and double selection, plants were raised that significantly exceeded the original specimen in terms of fruit set under conditions of elevated temperatures. Based on the available literature data, we can say that in tomatoes in the phase of mature pollen grain there is an opportunity for selection at the haploid level in order to change the tolerance to temperature stress of sporophytic offspring [28, 29].

Microgametophytic selection requires an answer to the question of the duration and magnitude of the effect on pollen in order to ensure both a sufficient strength of selection and the formation of an acceptable number of seeds in fruits. The material for the study was pollen of interspecific tomato hybrids obtained from the crossing of the Mo 500 mutant line with the wild species Lycopersicum minutum Rick and Solanum pennellii Corr. The pollen was heated in a thermostat at 58°C for 1, 2, 3, 6, and 12 h.

As a result of this experimental study, it was found that 6 and 12 h of heat treatment of pollen completely deprived it of the ability to germinate. Fruits were not set during pollination with such pollen, which indicated the loss of not only vitality but fertilizing ability as well. As optimal for gametophytic selection a 3 h treatment was accepted, which reduced the viability of pollen from 20–30% to 2–5%. The sum of temperatures under this effect amounted to 174°C and was close to the sum of lethal temperatures found for tomato varieties with high heat resistance of pollen.

Subsequently, the pollen of interspecific hybrids after 1 and 3 h of heating was used for pollination and raising F₂ and BC₁ offspring. It should be noted that the Mo 500 inbred line is a multimarket mutant, which is marked with 4 genes located in the 2nd and 6th chromosomes. These genes are easily identified in the early stages of plant development.

Marker analysis of F₂ and BC₁ segregating plant populations revealed differences after pollen treatment. Deviations from the control were insignificant for genes marking the second chromosome but significant for gene “c” (potato leaf), localized on the 6th chromosome (Table 7).

Pollen treatment	F2 phenotypes			Segregation Ratio
	Normal plants	Plants with marker-trait	Total
F2 Mo 500 × L.minutum
Fresh pollen (control)	676	114	790	5.93:1
Pollen heated at 58°C/1 h	514	86	600	5.98:1
Pollen heated at 58°C/3 h	521	71	592	7.34:1*
F2 Mo 500 × S.pennellii
Fresh pollen (control)	342	84	426	4.07:1
Pollen heated at 58°C/1 h	348	84	432	4.14:1
Pollen heated at 58°C/3 h	370	70	440	5.29:1*
BC1 Mo 500 (Mo 500 × S.pennellii)
Fresh pollen (control)	140	161	301	0.87:1
Pollen heated at 58°C/3 h	196	154	350	1.27:1*

Table 7.

Influence of pollen heating of F₁ tomato hybrids on the segregation ratio in F₂ or BC₁ generations for “potato leaf” marker-trait.

Note: χ²_0,05 (df = 1) = 3.84; * differences from the control are significant at the 0.05 level of probability.

Only a 3 h treatment of F₁ hybrid pollen with high temperature led to a deviation of monogenic ratios in the direction of increasing the number of alleles of wild species. A decrease in the number of recessives at the C locus, emerged as a result of a 3 h temperature treatment of the hybrid pollen indicates that selection for heat resistance was accompanied by a predominant elimination of pollen grains of mutant type as compared to wild ones, such as L. minutum and S. pennellii.

In general, of the 4 studied loci, only the C locus has proved to be “temperature-sensitive” at the stage of mature pollen. Later, marker loci of the 4th and 11th chromosomes were involved in research. However, analysis of F₂ and BC₁ plants after heating of F₁ hybrid pollen did not reveal significant differences from the control in the allele frequencies of wild tomato species for the marker genes from those chromosomes.

It can be assumed that the 6th chromosome of a tomato or its section within the immediate vicinity of the C locus determines to some extent the sensitivity of mature pollen to high temperatures. It is important to note that the same locus was also “perceptive” to low temperatures as it fluctuated in segregations in accordance with pollen germination ability and pollen tube growth at cold stress.

To reveal the differences between the segregating populations of sporophytes obtained after pollination with fresh and heated pollen, those populations were tested against the high-temperature background at the stage of seedlings and 4–6 leaves. It was determined that in both cases the experimental populations exceeded the control ones in terms of heat resistance. That is, pollen selection for high-temperature resistance is effective and can be used as a helpful tool in breeding programs.

2.5 Heat tolerance of pollen and gametophytic selection for resistance to temperature stress in maize

Selection for sporophyte tolerance to high temperature and drought is a part of many maizes breeding programs. But there is little information on the heat sensitivity of reproductive processes and structures, specifically pollen. At the same time, it is known that maize pollen is characterized by a low ability to resist adverse environmental conditions. High temperature combined with low relative air humidity does have the most destructive effect.

2.5.1 Effects of high temperature on maize mature pollen grains

Pollen of maize inbred lines of various origins were collected on sunny days. Then it was placed on glass slides (without medium) in one pollen grain layer. Some slides were placed into a thermostat and heated in the dark at 35°C for 3, 5, 10, and 20 min and other slides were treated for 10, 20, and 30 min at 26°C. After the heat treatment, pollen was inoculated on the nutrient medium. Fresh pollen was used as the control. The percentage of pollen grain germination and pollen tube length were scored. Tolerance of pollen to high temperature was defined by the decrease in viability as compared to the control.

Effects of temperature on the percentage of pollen grain germination in two contrast maize inbreeds (A641 and MK386 lines) are presented in Table 8.

Heating temperature, °C	Heating exposition, min	A641	MK386
—	Control	45.9 ± 2.2	89.1 ± 1.3
26	10	45.6 ± 2.2	4.2 ± 0.8*
26	20	25.3 ± 1.8*	0*
26	30	18.2 ± 1.7*	0*
35	3	40.8 ± 2.1	9.3 ± 1.1*
35	5	45.3 ± 2.1	0.8 ± 0.3*
35	10	13.4 ± 1.5*	0*
35	20	1.0 ± 0.5*	0*

Table 8.

Effects of mature pollen heating on pollen grain germination percentage in two maize accessions with contrasting heat resistance.

Note: * differences from the control are significant at the 0.001 level of probability.

Heating mature pollen at 35°C decreased maize mature pollen grain viability even at very short exposures (3–5 min) in MK386 inbred but such treatments did not affect germination percentage in A641 inbred line. Longer treatments (10 min and more) considerably decreased pollen grain germination in A641 and completely inhibited the germination process in the MK386 line. In contrast to the MK386 line, the pollen of A641 inbred endured heating at 35°C for 20 min. Pollen of these accessions also lost its viability at 26°C. After 30 min treatment, MK386 pollen perished completely, while at the same exposure A641 pollen was characterized by the sufficiently large number of germinated pollen grains.

Differences in the tolerance of maize mature pollen to high temperatures were revealed among the inbreeds both at 26°C and 35°C. Even a 5-min treatment at 35°C resulted in decreases in pollen viability that varied in different inbreeds, as compared to the control, from 1.3% in A641 line to 99.1% in MK386 inbred. The effects of high temperatures on pollen tube length were similar. The data obtained allowed us to conclude that the genotypes with high-temperature resistant pollen were characterized not only by higher germination percentage, but also a better ability to develop normal pollen tubes at high temperatures.

The mechanism which induces the rapid loss of viability of tricellulate cereal pollen is not yet clear. A reduction up to 80% of the original water content of maize pollen did not essentially affect its viability. With water loss greater than this value, pollen grains undergo irreversible changes.

A comparison of the heat resistance of pollen of inbred lines with their heat resistance at the stage of 20-day-old seedlings suggests a certain pattern. Lines with pollen more resistant to high temperature are also characterized by more resistant to this stress sporophyte. The correlation coefficient between the temperature resistance of the male gametophyte and the sporophyte turned out to be rather high and varied in different years from 0.6 to 0.78.

The positive relationship between the resistance to high temperatures of the gametophyte and sporophyte suggests that this trait is controlled by the same genetic system at both the haploid and diploid levels. In this case, the assessment of the sporophyte quality can be made based on the analysis of pollen traits.

Genotypic differences in the response of pollen to temperature stress give rise to the hope that the selection of mature pollen grains in a heterogeneous population will be successful in increasing the resistance of the sporophytic generation to high temperatures. In maize, mature pollen can be not only a tool for increasing sporophyte resistance. Mature pollen can be a breeding goal, given that the resistance of pollen grains to high temperatures after they are shed from the anthers is no less important for ensuring high crop yields than the resistance of a vegetative plant.

2.5.2 Effect of selection of pollen resistant to high temperature in F₁ on drought tolerance of F₂ sporophytes in maize

The effectiveness of gametophytic selection for drought resistance in maize can be demonstrated using the example of the VIR27 × MK01 hybrid. Its parental components were contrasting in resistance to high temperatures at the haploid and diploid stages of development.

To carry out the selection, the pollen of the hybrid was heated at the temperature of 35°C for 5–20 min. After heating, that pollen was used for self-pollination of the hybrid, having previously determined its viability on an artificial nutrient medium. For pollination, the pollen was used, which reduced its viability after heating by at least 80%. Pollination of each ear was carried out with a limited amount of pollen in order to exclude competition between haploid genotypes during pollen germination and pollen tube growth. Seeds (F₂ sporophytes) obtained after pollination of F₁ plant with fresh (control) and heated (treatment) pollen were sprouted in a solution with an osmotic (18 atm) and after a certain time, the percentage of germinated kernels was calculated.

As a result of heating the heterogeneous pollen population of this hybrid, the number of germinated seeds after 8 and 12 days under conditions of osmotic stress was 1.5 and 7.0 times higher than in the control. The data obtained allow to conclude about high efficiency of selection for drought resistance at the stage of mature pollen in maize.

High-temperature conditions when exposed to pollen are inevitably accompanied by low air humidity. Obviously, different reactions of haploid genotypes to high temperatures are due to different states of the membrane of pollen grains in genetically different haploid genotypes. Pollen grains resistant to these thermal treatments are characterized by such a membrane complex, which allows better water retention. As a result, such pollen retains its viability longer.

2.5.3 Changes in monogenic ratios for marker genes in F₂ as a result of high-temperature treatment of pollen from F₁ hybrids

A shift in Mendelian segregation ratios is one of the convincing evidence of the genetic activity of gametes. Segregation distortion for any marker gene as a result of treatment of a heterogeneous population of pollen is not yet evidence that this particular gene is expressed in the male gametophyte. A shift in the monogenic ratio for the marker locus can also be due to its linkage with the gene that determines the sensitivity of the gametophyte to the used selection agent. At the same time, conclusions about the localization of this gene (s) on a particular chromosome (chromosomes) of the genome can be drawn.

F₁ hybrids whose parent components were characterized by differences in heat tolerance at both gametophytic and sporophytic levels were used in our experiments. Mangelsdorf tester was one of the parents of those hybrids. This line is marked with 10 recessive genes, localized in different chromosomes. Seven markers were analyzed, which are typically expressed at the early stages of plant development: bm2 (chromosome (Chr) l), lg1 (Chr 2), a1 (Chr 3), su1 (Chr 4), gl1 (Chr 7), j1 (Chr 8), g1 (Chr 10).

Mature pollen grains of hybrids and pollen grains at the stage of their maturation were subjected to high-temperature treatment. In the latter case, maize tassels, which were transferred to a laboratory at the beginning of flowering, were exposed to temperature stress. After heat treatment, the pollen was used for self-pollination of the hybrids. In the control pollination was performed with fresh pollen.

With the help of marker analysis, the changes in segregation for seven recessive marker genes were evaluated in comparison with the control. For each F₂ population at least 400 plants were analyzed. In each case, the percentage of seed germination was quite high and amounted to more than 95%.

Pollen treatments of F₁ hybrids considerably influenced the monogenic ratio for some marker genes studied in the F₂ populations. The changes in dominant and recessive genotype ratios were found both after the treatment of mature pollen and pollen during its maturation. Following high-temperature treatment of pollen grains at the maturation stage, differences in the segregation for three markers loci—Gl₁, A₁, and Su₁ were observed. On the other hand, the treatment of mature pollen of F₁ hybrids led to a change in monogenic ratios for four marker loci on the first, third, fourth, and tenth chromosomes in comparison with the control (Table 9).

Male gametophyte stage	Temperature
	Low	High
Pollen maturation	a1 (3-th chromosome); su1 (4-th chromosome); g1 (10-th chromosome)	a1 (3-th chromosome); su1 (4-th chromosome); gl1 (2-th chromosome)
Mature pollen grain	bm2 (1-th chromosome); a1 (3-th chromosome); su1 (4-th chromosome); g1 (10-th chromosome)	bm2 (1-th chromosome); a1 (3-th chromosome); su1 (4-th chromosome); g1 (10-th chromosome)
Pollen germination and pollen tube growth	bm2 (1-th chromosome); su1 (4-th chromosome); lg1 (7-th chromosome); j1 (8-th chromosome)	–

Table 9.

Marker genes for which a shift in monohybrid ratios in F₂ was found as a result of temperature treatment of male gametophyte of F₁ hybrids of maize.

Comparing the data on changes in monohybrid ratios in F₂ as a result of exposure of the microgametophyte to high and low temperatures at the stages of pollen maturation, mature pollen grains and during germination and growth of the pollen tube, one can conclude that the direction of those changes largely depends on the stage of the male gametophyte than on the temperature. The male gametophyte of maize at each stage is characterized by its own set of expressed genes. Some of them, apparently, are expressed at the stages of development and functioning of the microgametophyte.

The presented data on the change in Mendelian segregation in F₂ under the influence of high temperature on the pollen of F₁ hybrids indicate the selectivity of this factor and the possibility of efficient selection of genotypes resistant to temperature stress at the microgametophytic level in maize.

3. Conclusions

Losses in crop yield caused by unfavorable abiotic factors, in particular drought, may reach large dimensions. Drought is usually accompanied by temperature spikes which invoke damage of plants and decrease the yield. Therefore, increasing the drought and heat tolerance are important directions in the modern breeding of cultivated plants.

Along with the traditional methods of selection for tolerance to abiotic stresses at the level of seeds, seedlings, or plants the methods of gametophytic selection, including pollen selection, can be successfully used. It was established that a large part of the genes expressed in the pollen are also exhibits in the plant. This fact indicates that gene transcription occurs in the haploid genome and therefore selection of the traits, which are under the control of genes expressed at both plant and microgametophyte (pollen) levels can be performed effectively.

In our research with sunflowers, when freshly collected pollen of F₁ plant was heated and used for self-pollination, the drought resistance of the next F₂ generation was significantly enhanced. This was confirmed by the large number of plants that can survive to bloom in dry field conditions as well as better F₂ seed germination in osmotic solutions. In another experiment, germination of sunflower pollen in vivo under osmotic stress with polyethylene glycol solution as the selective barrier increased seed germination on the osmotic medium in vitro.

High efficiency of the pollen selection technique for drought tolerance was revealed by us earlier in other crops, such as tomato, maize, castor bean, and linseed. It was in this way that the flax variety was obtained which is a National Standard in Ukraine, and is perfectly adapted to the dry conditions of the south of the country.

Evaluation at the level of pollen may also be of independent interest since the sensitivity to a drought of the plant reproductive system itself is a very important characteristic for many agricultural crops. A possibility to use this approach at the earliest stages of the breeding process, especially when the breeding material is quantitatively limited, is an undeniable advantage of the proposed assessment method.

In general, in the suggested chapter, using as an example different agricultural crop, the effectiveness of pollen selection techniques for drought resistance were demonstrated, as well as methods for assessing plant drought tolerance by its male gametophyte.